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Midi Prep
The QIAGEN Midi Prep allows us to purify and Isolate Bacterial DNA. Mark tends to use the QIAGEN-100 tip DAY 1: Pick a single colony from a freshly streaked selective plate (eg. LB+AMP) and inoculate a stater culture of 2-5 mL of LB containing the appropriate selected antibiotic -Incubate for 8 hours at 37 degrees C with vigorous shaking. DAY 2: 'Dilute the starter culture from 1/500 to 1/1000 into selective LB medium ''FOR HIGH COPY PLASMIDS: Inoculate 25 mL with 25-50 uL of starter culture(QIAGEN-tip 100) or 100mL medium with 100-200uL of starter culture(QIAGEN-tip 500). FOR LOW COPY PLASMIDS: Inoculate 100 mL with 100-200 uL of starter culture(QIAGEN-tip 100) or 500mL medium with 250-500uL of starter culture(QIAGEN-tip 500). Grow at 37 degrees Celsius for 12-16 hours with vigorous shaking. IMPORTANT!: Remember to set your Centrifuge temperature to 4 Degrees Celsius the night before you plan to harvest your bacteria. '''DAY 3: 1. Transfer your Bacterial broth from your 250 mL Erlenmeyer flasks into the correct volume of centrifuge tube (50 mL) 2. Harvest your Bacterial cells by centrifuging at 2500 RPM for 15 min at 4 degrees Celsius. 3. Decant the supernatant and re-suspend the bacterial pellet in 4 mL (QIAGEN-100 tip) or 10mL(QIAGEN-500 tip) in BUFFER P1, by sucking up and spitting back out the buffer until the pellet breaks up, and lightly vortexing -Shake the bottle if it has LyseBlue Reactant. 4. Add 4mL (QIAGEN-100 tip) or 10mL (QIAGEN-500 tip) of BUFFER P2. Mix vigorously by inverting the sealed tube 4-6 times, and incubate at room temp (15-25 degrees C) for 5 Min. -Don't vortex as it will shear the DNA -lysate should be viscus, Don't allow it to sit for more then 5 min. -Cover the Buffer immediately to avoid acidification from CO2 5. Add 4mL (QIAGEN-100 tip) or 10mL(QIAGEN-500 tip) of Chilled P3 Buffer, Mix immediately and thoroughly by vigorusly inverting the sealed tube 4-6 times, incubate on ice for 15 min(QIAGEN-100 tip) or 20 min (QIAGEN-500 tip). -a white fluff will form -the precipitate will contain DNA, Proteins and Cellular debris. 6. Centrifuge at 11,000RPM for 1hr. 7. Move the supernatant to new labled tube and Centrifuge again at 11,000 RPM for 30 min at 4 degrees C, remove supernatant and plasmid promptly. 8. Equilibrate a column by applying 4mL(QIAGEN-100 tip) or 10mL (QIAGEN-500 tip) BUFFER QBT, and allow the column to empty by gravity flow. Place the column hanging on top of a new tube 9. Apply the Supernatant from step 8 to the Column and allow it to enter the resin by gravity flow. 10. EMPTY THE Tube into a waste container and place the now empty tube back under the column. 11. Wash the column with 10mL (QIAGEN-100 tip) or 30mL (QIAGEN-500 tip) twice with BUFFER QC 11b. Now Replace the tube with a New labled tube. 12. Elute the DNA with 5mL (QIAGEN-100 tip) or 15mL (QIAGEN-500 tip) BUFFER QF 13. Precipitate the DNA by adding 3.5 mL (QIAGEN-100 tip) or 10.5 (QIAGEN-500 tip) of ice cooled ISOPROPANOL to the eluted DNA and Centrifuge at 11,000 RPM at 4 degrees C for 1 hour. -IF YOU RUN OUT OF TIME PLACE THE DNA WITH ISOPROPANOL IN THE -20 FREEZER BEFORE CENTRIFUGING! 14. Remove the Isopropanol. 15. Add 2mL 70% Ethanol by pipeting on the opposite side of the pellet 16. Spin at 11,000 RPM for 15 min at 4 degrees C. 17. Remove the Ethanol and dry by carefully pouring out the tubes onto paper towels and letting them sit upside-down for five minutes on the paper towels. 18. Re-suspend Pellet in 300uL dH2O, Swirl and transfer to microcentrifuge tubes. 19. Spec via nanodrop, enter into the spreadsheet number and freeze.